Troponin I (TnI) is a component of a heterotrimeric complex, along with troponin C (TnC) and troponin T (TnT), involved in regulation of vertebrate striated muscle contraction (Zot and Potter, Annu. Rev. Biophys. Biophys. Chem. 1987, 16:535-559; Farah and Reinah, FASEB J. 1995, 9:755-767). Muscle contraction is triggered by the binding of Cam−+ ions to TnC. TnT binds to tropomyosin anchoring the Tn to the muscle filament. TnI is the inhibitory subunit of the troponin complex, binding to actin-tropomyosin complexes and preventing the interaction of actin and myosin. TnI is present in muscle tissue in multiple isoforms expressed from a multi-gene family (Wu et al., DNA Seq. J. DNA Seq. Mapp. 1993, 4:113-121).
Recent investigations have demonstrated a second important biological function for TnI, the ability of the molecule to inhibit both in vitro endothelial cell survival/proliferation and inhibit in vivo angiogenesis (the growth and development of blood vessels) (Moses et al., Proc. Natl. Acad. Sci. USA 1999, 96:2645-50). TnI that inhibited endothelial cell development was isolated initially from cartilagenous tissue, and subsequent studies demonstrated anti-angiogenic activity with recombinant forms of TnI expressed in E. coli. 
The TnI subunit is a single polypeptide with a molecular weight of 21,338. The molecule contains three cysteine residues, at positions 45, 65, and 134 (Wilkinson and Grand, Biochem J. 1975, 149:493-496). Purification processes previously developed to isolate native TnI from tissue sources or recombinant TnI require maintenance of a reducing environment throughout the purification and storage of TnI, ordinarily by the addition of dithiothreitol (DTT) to protein preparations (Potter, Methods Enzymol., 1982, 85:241-263; Jha et al., Protein Exp. Purif., 1994, 5:604-613; Al-Hillawi et al., Eur. J. Biochem., 1994, 225:1195-1201). The added DTT maintains the TnI cysteine sidechain sulfhydryls in their reduced state, preventing the formation of intra- or intermolecular disulfide bond crosslinks. Disulfide bonds between TnI cysteine sidechains are not believed to be present in the active conformation of the protein involved in regulation of muscle contraction (Kluwe et al., FEBS Lett., 1993, 323:83-88), and reductant was utilized in the isolation of active anti-angiogenic forms of TnI.
The necessity of maintaining a reducing environment in processing and storage during large scale purification of TnI for commercial use poses numerous technical difficulties and increases production cost. Thus, there is a need in the art to purify and refold Tropinin I efficiently and affordably.